ANDERSSON, Karl (Ulleråkersvägen 62, Uppsala, S-756 43, SE)
| CLAIMS:
1. A method for the measurement of cell status, comprising:
attaching target cells to be studied to a selected portion of a solid support;
attaching a cell status marker to the cells; incubating the cells in a solution of a suitable medium;
treating the cells to bring about the condition, the effect on cell status of which is to be studied;
performing a measurement, capable of detecting presence of the status marker on or in the target cells;
reducing the amount of solution covering the selected portion of the support prior to performing the measurement;
making a reference measurement on a portion of the support where no target cells are present.
2. Method as claimed in claim 1 , wherein said status marker is an emitter of radioactive radiation.
3. Method as claimed in claim 1, wherein said status marker is inherently fluorescent or labelled with a fluorescent moiety.
4. The method as claimed in claim 1 or 2 or 3, wherein a difference between target and reference measurements is calculated.
5. The method as claimed in any of claims 1-4, wherein the solid support is an essentially flat dish capable of holding a solution confined within its boundaries.
6. The method as claimed in any of claims 1-5, wherein the status measured is viability and the viability marker is 51 -chromium.
7. The method as claimed in any of claims 1-5, wherein the status measured is viability and the viability marker is any of Calcein- acetoxymethyl ester diacetate; 5-Carboxyfluorescein; 5-Carboxyfluorescein Diacetate; 3,3'-Dihexyloxacarbocyanine Iodide; DiOC2(5); 3,3'- diethyloxadicarbo- cyanine iodide; or Rhodamine 123.
8. The method as claimed in any of claims 1-5, wherein the status measured is viability and the viability marker is a chemoluminiscent molecule.
9. The method as claimed in any of claims 1-8, wherein the reduction of the amount of solution is achieved by orienting the support at an angle that deviates from the horizontal to provide an elevated part and a lower part of said support, such that the elevated part will be covered by less solution than the lower part, and wherein the support is rotated at a predetermined speed of rotation.
10. A method for the measurement of cell status, comprising the following steps:
attaching target cells to be studied to a selected portion of a solid support;
attaching a cell status marker to the cells; incubating the cells in a solution of a suitable medium, and optionally performing a washing step;
treating the cells to bring about the condition, the effect on cell status of which is to be studied, and optionally washing the treated cells;
said attaching and treating steps optionally being performed simultaneously, or in reversed order, and optionally being followed by a washing step;
performing a measurement, capable of detecting presence of the status marker on or in the target cells;
reducing the amount of solution covering the selected portion of the support prior to performing the measurement;
making a reference measurement on a portion of the support where no target cells are present. |
METHOD FOR MEASUREMENT OF CELL VIABILITY
FIELD OF INVENTION
The present invention relates to the field of biological research. More in particular, it relates to biological research utilizing living cells. Even more in particular, it relates to the measurement of the status of cells, such as the viability of cells.
BACKGROUND OF THE INVENTION Biological research of today is to a significant fraction relying on cells as model organisms. In many cases, the measurement of how cell status is dependant of a certain treatment is of great interest. Cell status may include qualities as cell growth, changes in cell morphology, triggering of repair mechanisms after damage, viability, and the like. One common example of cell status measurement is the viability measurement, i.e. the measurement of the ability of a cell to survive a certain treatment is of great interest. The reverse is also common, i.e. the measurement of how efficient a certain treatment is in killing cells. Yet another viability measurement of interest is to follow natural cell death (apoptosis) either through triggering of apoptotic molecular pathways (i.e. not poisoning the cell, but rather trick it to commit suicide) or as it occurs without use of molecular effectors.
One particular area in which cell status measurements, and in particular viability measurements, is common is immunology, where the efficiency and function of natural killer cells are of interest. Natural killer cells are of vital importance in diseases targeting the immune system, such as HIV, as described by Nair MP, Saravolatz LD, and Schwartz SA in "Selective inhibitory effects of stress hormones on natural killer (NK) cell activity of lymphocytes from AIDS patients" (Immunol Invest. 1995 Aug;24(5): 689-99), which is incorporated by reference herein. Thus, there is a need for methods capable of measuring cell viability. Since the results from different types of viability assays may not agree (as shown by
Langhans B, Ahrendt M, Nattermann J, Sauerbruch T, and Spengler U in "Comparative study of NK cell- mediated cytotoxicity using radioactive and flow cytometric cytotoxicity assays." (J Immunol Methods. 2005 Nov 30;306(l-2): 161-8.), which is incorporated by reference herein), there is a need for further refinement of the methods capable of measuring cell viability.
CLOSEST PRIOR ART
There are different methods available for the measurement of cell status. One common method for determination of DNA content in cells is by staining the cells with propidium iodide (PI) as described by Bruce Hudson and co-authors in "The use of an ethidium analogue in the dye-buoyant density procedure for the isolation of closed circular DNA: The variation of the superhelix density of mitochondrial DNA" (PNAS 62(3): 813-820 1969), which is incorporated by reference herein. Since PI bind stoichiometrically to DNA, the amount of detected PI is directly proportional to the DNA content and dividing cells can easily be differed from normal cells.
There are other methods available for the measurement of viability (such methods are sometimes referred to as toxicity measurements using the antonym toxicity). One common method relies on radioactive chromium (51Cr), which is absorbed by living cells and released when the cells die. Such measurements typically comprise an incubation period, during which the cells are loaded with 51Cr, a toxin treatment period (and optionally a waiting period) prior to harvesting the cell culture medium. Since dead cells have release their chromium into the cell culture medium, the radioactivity of the medium is proportional to the number of killed cells. Measurements relying on 51Cr are terminal and can therefore only provide information on the cell viability status at the time of harvesting. A more detailed description of the use of 51Cr is found in "Impaired NK cell cytotoxicity by high level of interferon-gamma in concanavalin A-induced hepatitis." (by Dong Z, Zhang
C, Wei H, Sun R, and Tian Z in Can J Physiol Pharmacol. 2005;83(l 1): 1045- 53.), which is incorporated by reference herein.
It is also possible to measure cell viability using non-radioactive methods, as described in "Comparative study of NK cell-mediated cytotoxicity using radioactive and flow cytometric cytotoxicity assays." (by Langhans B, Ahrendt M, Nattermann J, Sauerbruch T, and Spengler U in J Immunol Methods. 2005 Nov 30;306(l-2): 161-8.), which is incorporated by reference herein.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an improved method for measuring cell status in general. The invention is particularly useful for time-resolved measurements of cell viability. The method according to the invention is defined in claim 1.
In a preferred embodiment, the method for measuring cell viability uses of
51Cr.
The invention in a preferred embodiment comprises three characteristic components.
• A marker suitable for cell status,
• Cells for which the status shall be measured,
• A device suitable for the time-resolved measurement of the quantity of said marker that is associated with said cells.
Suitable markers include (but are not limited to) radioactive markers, fluorescent markers and chemoluminiscent markers. Suitable cells include (but are not limited to) prokaryotic or eukaryotic cells that can be cultured in a laboratory. One typical example of suitable cells is human cancer cell- lines, for example the cell-line A431 (ATCC, CLR 1555, Rocksville MD USA).
The time resolved measurement is typically made in a device described in WO2005080967, which is incorporated by reference herein.
There are numerous applications of the invention. One is the measurement of cell viability under the influence of poisonous compounds, for identifying robust cell-lines, or for identifying powerful toxins, or to estimate the danger associated with handling of novel compounds and chemicals. Another application is the study of apoptosis and potential apoptotic inducers and apoptotic inhibitors. Yet another application is the development of cancer treatments, i.e. finding suitable means for killing tumor cells. Still another application is the study of cell viability when put under influence of external treatments, such as cell resistance to electromagnetic radiation. Yet another application is to study the function or effect of the immune system when killing intruding cells. Still another application is the measurement of total DNA in the target cells as an indirect measure of growth.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be disclosed in closer detail in the description and example below, with reference to the accompanying drawing, in which
Figure 1 shows a flow chart of the method
Figure 2 shows a suitable instrument, known in prior art, for performing the measurement in the viability method; Figure 3 shows results from one 51Cr incubation experiment;
Figure 4 shows results from two 51Cr incubations on untreated cells and from one 51Cr incubation on treated cells;
Figure 5 shows results from two 51Cr incubations on untreated cells, wherein the 51Cr concentration was different; and Figure 6 shows results from a series of combined 51Cr incubations and treatments.
DETAILED DESCRIPTION OF THE INVENTION
For the purpose of the present application, and for clarity, the term "cell status" refers to the general condition of a cell population with respect to one of more aspects including (but not limited to) DNA content, intracellular metal ions concentrations, intensity of metabolism, fraction necrotic cells, and the like. The term "viability" refers to the viability status of the cells subject to measurement. Viable cells are alive and growing, and poor viability is recognized as slow or absent growth, poor cell morphology, or necrosis. The cells attached to the solid support are denoted "target cells". Target cells include (but are not limited to) pro kary otic cells, eukaryotic cells, tissue slices (thinner than 2 mm), and small organisms (less than 2mm in diameter) that can be cultured in a laboratory. More particularly, target cells include (but are not limited to) human cancer cell lines, human embryonic stem cell lines (naϊve or differentiated), cancer cell lines from other mammals, embryonic cell lines from other mammals, insect cells and xenopus cells. The method requires a compound to be used for monitoring the status of the target cells, said compound being denoted "status marker". Possible status markers include, but are not limited to, radioactive markers (e.g. 51Cr, 18-F-deoxyglucose), fluorescent markers (e.g. Calcein- acetoxymethyl ester diacetate; 5-Carboxyfluorescein; 5-Carboxyfluorescein Diacetate; 3,3'-Dihexyloxacarbocyanine Iodide; DiOC2(5); 3,3'- diethyloxadicarbo- cyanine iodide; Rhodamine 123) and chemoluminiscent markers (e.g. {2-(p-Hydroxybenzyl)-6- (p-hydroxyphenyl)-8-benzyl- imidazo[l,2-a]pyrazin-3-(7H)-one}).
The present invention aims at providing a method of measuring cell status by enabling a time-resolved detection of status markers in target cells. Due to the time-resolved detection, the effect of different chemical or environmental conditions can be detected as it appears.
Some of the possible implementations of the method used for cell status measurement are outlined in figure Ia. In a first implementation, target cells
are loaded with viability markers (110), followed by an optional wash (120). Next, the cells are treated with the agent or exposed to a condition for which the status should be determined (130), followed by an optional wash (140). The last step in the method is the time-resolved detection of status marker associated with target cells (150), which is a direct or indirect measure of the status of the cells.
There are a variety of conditions that the target cells can be exposed to. Possible agents include (but are not limited to) drugs-like compounds, for example cytotoxic drugs (e.g. Doxorubicin, Daunorubicin, Fluorouracil, Tiotepa, Hydroxykarbamid, Karboplatin, Metotrexat, Etoposid, Paklitaxel, Vinkristin, Dakarbazin, Epirubicin, Oxaliplatin, Lomustin, Cytarabin, Vinorelbin, Docetaxel, Pemetrexed, Topotekan, Amsakrin, Merkaptopurin, Busulfan, Melfalan, Metotrexat, Idarubicin, Mitoxantron, Mitomycin, or Capecitabin) . Other conditions include, but are not limited to, cytotoxic proteins (e.g. monoclonal antibodies like Rituximab, Trastuzumab, and Cetuximab), growth factors (e.g. EGF, NGF, TGF-alpha, TGF-beta), other proteins (e.g. insulin), detergents (e.g. tween 40, tween 20, Triton XlOO, octylglycoside or NP40), inorganic compounds or metals (e.g. Copper, Lead, Cadmium, or Mercury), radioactive compounds (e.g. 123-iodine, 131-iodine, 125-iodine, 177-lutetium, 213-bismuth, 211 -astatine, 225-actinium, 11- carbon, 18-fluorine, 99-tecnetium, 32-phosphorus, 33-phosphorus, 35- sulfur, either as free nuclides or synthesized into a compound) and the like. Possible conditions include (but are not limited to) starvation, hyperthermia, hypothermia, increased or decreased ionic strength compared to physiological conditions, increased or decreased pH compared to physiological conditions, non-ionizing radiation, ionizing radiation, static electromagnetic fields, fluctuating electromagnetic fields (e.g. microwave treatment), and mechanical treatment (e.g. ultrasound treatment or centrifugation) . In measurements of apoptosis, a range of apoptosis- inducing effector compounds can be used, including (but not limited to) bisindolylmaleimide compounds (as defined in US6284783, which is
incorporated by reference herein), butylated hydroxyanisole, allyl sulfide, benzyl isothiocyanate, and dimethyl fumarate.
The method can be conducted in slightly different ways. One other example is as described in figure Ib, wherein the treatment of the target cells could be the first step (111), followed by an optional wash (121), after which the treated target cells are loaded with status marker (131). After an optional wash (141), status marker is detected using a time-resolved detection technology.
Yet another example is as described in figure Ic, wherein the treatment of the target cells and the loading of status marker is performed simultaneously (112), followed by an optional wash (122). Next, status marker is detected using a time-resolved detection technology (132).
Still another example is as described in figure Id, wherein the first step is the treatment of the target cells (113), followed by an optional wash (123). Next, status marker is loaded and simultaneously detected using a time- resolved detection technology (133).
Yet another example is as described in figure Ie, wherein the first step is the loading of status marker during time- resolved detection (114), followed by an optional wash (124). Next, the treatment of the cells is started and the effect of it being simultaneously detected using a time-resolved detection technology (134).
There are several methods available for the time-resolved measurement of viability marker in target cells. The preferred method for completing steps 114, 150, 151, 132, 133, and 134 in figure 1, i.e. the detection, has been previously disclosed [WO2005080967, which is incorporated by reference herein] and is schematically described in figure 2. In brief, the method relies on target cells (202) being attached to a defined area on a solid support
(201). On the same solid support, there is also a reference area (in this case opposite to the target area). A liquid is in contact with the solid support to enable a suitable environment for the target cells. The liquid may possibly contain status marker, so that the loading of target cells takes place during detection. Furthermore, the solid support is inclined and slowly rotated using a motor (203). A detector capable of detecting the label attached to the species used is mounted (204) over the elevated portion of the solid support. When the target area passes the detector, an elevated signal will be registered in case the status marker has bound to or is present in the target cells. The rate of change of status marker density can be followed by depicting the difference between the detected signal from target area and reference area over time.
The generic properties of the preferred detection method using the apparatus shown in figure 2 are the following:
• target cells are attached to a selected portion of a solid support,
• the target cells are exposed to a solution, possibly containing status marker,
• a measurement is performed, capable of detecting presence of status marker on or in target cells, during which the amount of solution covering the selected portion of the support is reduced, and
• a reference measurement is performed on a portion of said support.
In summary, the present invention makes it possible to follow cell status in real time, thereby monitoring the progress of processes toxic to the cell.
The following non-limiting examples of the invention will illustrate the principle behind it.
EXAMPLE Example 1
The method described above (figure Id, steps 113- 123- 133) was tested with target cells of type A431 (a human cancer cell-line) grown on approximately one quarter of a 10cm circular cell-dish. The treatment comprised incubation of the cells with a DNA-interchelating compound labeled with 125-iodine denoted "Agent". The Agent is known to be cytotoxic. The cellular status detected in this example was viability and the status marker was 51- chromium. In order to establish a baseline, one measurement of cell viability without treatment was performed. Thus, step 113 and 123 were ignored and the baseline measurement started at step 133. A time-resolved measurement was performed using the preferred device as described in figure 2, using a detector sensitive to gamma radiation in the energy range 100- 1000 keV. The resulting graph of amount of 51Cr versus time is shown in figure 3. As seen, the cells absorb 51Cr during approximately 16 hours after which the 51Cr is declining. The baseline measurement was repeated in order to verify the stability of the method.
Next, target cells were treated with Agent during one hour (113), followed by a wash (123) and approximately 15 hours after Agent treatment, the target cells were loaded with 51Cr during measurement (133). The resulting curve of amount of 51Cr versus time is shown in figure 4, together with the two baseline curves. Figure 4 shows that the decline of 51Cr is delayed in the treated cells compared to the baseline measurement. Furthermore, the results from the two baseline measurements are in agreement, indicating that the viability measurement is robust.
A probable cause for the general shape of the curves in figure 3 and figure 4 is that the target cells might be sensitive to 51Cr itself. The decline of 51Cr appeared earlier at higher concentration of 51Cr as shown in figure 4. The fact that the cytotoxic Agent is shifting the decline towards later points in
time indicates that Agent is forcing target cells into cell-cycle arrest, because dividing cells are likely more sensitive to metal poisoning than static cells.
Example 2 The method described above (figure 1, steps 114-124- 134) was tested with target cells of type A431 (a human cancer cell-line) grown on approximately one quarter of a 10cm circular cell-dish. The cellular status detected in this example was viability and the status marker was 51 -chromium. While detecting the 51Cr level in the cells, the cells were incubated with 51Cr during approximately 2h. After incubation, a toxic agent (Triton XlOO) was added to the cell dish. The release of 51Cr was then followed approximately 7 hours. The procedure was repeated several times with varying concentrations of the toxic agent Triton XlOO. Cellular 51Cr levels are displayed in figure 6, wherein the incubation of 51Cr is seen as a linear uptake, and the treatment time displays varying sensitivity to the toxic agent. It was concluded that at Triton XlOO greater than 0.03%, A431 cells were immediately killed as seen by the rapid release of 51Cr. At concentrations from 0.006% and downwards, the cells were essentially undisturbed as seen from the stable level of 51Cr over time. At concentrations between 0.006% and 0.03%, cell death occurred gradually over the course of many hours. All time-resolved measurements were performed using the preferred device as described in figure 2, using a detector sensitive to gamma radiation in the energy range 100- 1000 keV.
Although the invention has been described with regard to its preferred embodiments, which constitute the best mode currently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having ordinary skill in this art may be made without departing from the scope of the invention as set forth in the claims appended hereto.
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